Regulation of Ku70 methylation and functions by SETD4
SETD4 对 Ku70 甲基化和功能的调节
基本信息
- 批准号:10228239
- 负责人:
- 金额:$ 35.82万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-02-01 至 2026-01-31
- 项目状态:未结题
- 来源:
- 关键词:AcetylationAcinus organ componentAffectAllelesAnimal ModelAntibodiesApoptosisApoptoticBAX geneBax proteinBindingBiochemistryC-terminalCell NucleusCellsCellular biologyChromatinCytoplasmDNADNA DamageDNA Double Strand BreakDNA RepairDevelopmentDimerizationDouble Strand Break RepairElementsEnsureEnzymesEpitopesEventExcisionG22P1 geneGenomeGoalsHealthHistidineHistonesHumanHuman GenomeIonizing radiationKnock-in MouseKu70 proteinLoxP-flanked alleleLysineMalignant NeoplasmsMeasuresMediatingMethylationMethyltransferaseMitochondriaModelingMolecularMolecular BiologyMolecular ConformationMusNonhomologous DNA End JoiningNuclearOxygenasesPlantsPlayPositioning AttributeProteinsReagentRegulationRibulose-Bisphosphate CarboxylaseRoleS-AdenosylmethionineSET DomainSideSignal TransductionSiteStructureTertiary Protein StructureTestingTimeTransferaseXRCC5 geneantibody detectionbasedimerin vivoirradiationmethyl groupmouse geneticsnoveloverexpressionrecruitresponseribulose-1,5-bisphosphatesuccesstherapy outcometumortumorigenesis
项目摘要
Regulation of Ku70 Methylation and Functions by SETD4
(Abstract)
The objective of this study is to elucidate a novel regulatory mechanism of Ku70 functions that are controlled by
lysine methylation. Ku70 is a critical protein in DNA damage repair, especially during the initiation of non-
homologous end-joining after irradiation. This function is carried out by its dimerization with Ku80 and encircling
of DNA at the break sites. The free form of Ku70 is known for its anti-apoptosis activity in the cytoplasm, due to
its binding with the pro-apoptosis protein BAX. Our preliminary studies suggest that SETD4, a putative non-
histone methyl-transferase, methylated Ku70 to cause Ku70 relocation to the cytoplasm. Over-expression of
SETD4 suppressed apoptosis, while SETD4 depletion sensitized it. SETD4’s chromatin-binding was dependent
on Ku70, but not vice versa. SETD4 can be recruited to DNA damage sites, but only at a relatively mid-late time
point after DNA damage. Based on these novel findings, we hypothesize that Ku70 methylation by SETD4 plays
a critical role for the functional translocation of Ku70 from DNA double strand breaks (DSB) to the cytoplasm.
We have generated highly specific antibodies against methylated Ku70 and SETD4, and several Ku70 and
SETD4 knock-in mouse lines. We strive to use a combined approach that integrates biochemistry, cell and
molecular biology, and mouse genetics to test our hypothesis. In Aim 1, we will focus on Ku70-methylation and
its anti-apoptotic and DNA repair activities. First, the consequence of Ku70 methylation on Ku70/Ku80 dimer
stability and its binding to DNA will be determined. Second, the cytoplasmic activity of methylated Ku70 in
apoptosis will be verified with non-DNA damaging agents. Third, the direct effect of Ku70 methylation on Ku70
recruitment and retention at DNA damage sites, DSB repair efficiency, and cellular sensitivity to ionizing radiation
will be measured. Lastly, we will use in-house developed Ku70 knock-in mice to characterize the functions of the
lysine-containing SAP domain of Ku70 and its methylation in vivo. In Aim 2, we will focus on how SETD4
regulates apoptosis and DNA damage response through Ku70. First, we will identify the structural elements that
are critical for SETD4 to methylate Ku70. Second, the consequence of SETD4 modulation on apoptosis will be
measured in cells incapable of Ku70 methylation. Third, we predict that, while Ku70 is required for SETD4
recruitment to DNA damage sites, the SETD4’s enzymatic activity may be required for Ku70 disassociation from
DNA damage sites. Thus, the mutual roles of SETD4 and Ku70 on their recruitment and/or retention at DNA
damage sites will be determined, and their effects on DNA repair will be assessed. Lastly, we have tagged the
floxed mouse Setd4 allele with V5 and Flag (V5F) epitopes. We will use this mouse line to systematically analyze
SETD4’s role in Ku70 methylation, and its subsequent contributions in development and tumorigenesis. These
studies are expected to elucidate a previous unknown mechanism that coordinates Ku70 functions in the nucleus
and cytoplasm. The success of this project is ensured by our unique reagents and animal models as part of a
rigorous approach.
SETD4对Ku70甲基化及其功能的调控
(摘要)
本研究的目的是阐明Ku70功能的一种新的调节机制,该机制由
赖氨酸甲基化。Ku70是DNA损伤修复中的关键蛋白,特别是在非
照射后的同源末端连接。这一功能是通过其与Ku80的二聚化和环合来实现的
断裂点的DNA。游离型Ku70以其在细胞质中的抗凋亡活性而闻名,这是由于
它与促凋亡蛋白Bax结合。我们的初步研究表明,SETD4,一种推定的非
组蛋白甲基转移酶,使Ku70甲基化,导致Ku70重新定位到细胞质。过度表达
SETD4抑制细胞凋亡,而SETD4耗竭则使其敏化。SETD4‘S染色质结合依赖
在Ku70上,但反之亦然。SETD4可以被招募到DNA损伤部位,但只能在相对较晚的时间
指向DNA损伤。基于这些新的发现,我们假设由SETD4引起的Ku70甲基化起作用
Ku70在从DNA双链断裂(DSB)到细胞质的功能性转位中起着关键作用。
我们已经产生了针对甲基化Ku70和SETD4的高度特异性抗体,以及几种Ku70和
SETD4敲入小鼠品系。我们努力使用一种结合了生物化学、细胞和
分子生物学和老鼠遗传学来验证我们的假设。在目标1中,我们将重点介绍Ku70-甲基化和
其抗细胞凋亡和DNA修复活性。第一,Ku70甲基化对Ku70/Ku80二聚体的影响
稳定性及其与DNA的结合将被确定。第二,甲基化的Ku70在细胞质中的活性
细胞凋亡将用非DNA损伤剂进行验证。第三,Ku70甲基化对Ku70的直接影响
DNA损伤部位的募集和保留、DSB修复效率和细胞对电离辐射的敏感性
将会被测量。最后,我们将使用内部开发的Ku70敲入小鼠来表征
Ku70含赖氨酸SAP结构域及其体内甲基化在目标2中,我们将重点介绍SETD4如何
通过Ku70调节细胞凋亡和DNA损伤反应。首先,我们将确定以下结构元素
是SETD4甲基化Ku70的关键基因。其次,SETD4对细胞凋亡的调节作用将是
在不具备Ku70甲基化能力的细胞中进行测量。第三,我们预测,虽然SETD4需要Ku70
募集到DNA损伤部位,可能需要SETD4的S酶活性来解离Ku70
DNA损伤部位。因此,SETD4和Ku70在其在DNA中的招募和/或保留中的相互作用
将确定损伤位置,并评估其对DNA修复的影响。最后,我们已经标记了
带有V5和FLAG(V5F)表位的小鼠Setd4等位基因。我们将利用这条鼠标线来系统地分析
SetD4‘S在Ku70甲基化中的作用及其在肿瘤发生发展中的作用。这些
预计研究将阐明先前未知的协调核内Ku70功能的机制
和细胞质。该项目的成功是由我们独特的试剂和动物模型作为
严谨的方法。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Zhiyuan Shen其他文献
Zhiyuan Shen的其他文献
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{{ truncateString('Zhiyuan Shen', 18)}}的其他基金
Regulation of Ku70 methylation and functions by SETD4
SETD4 对 Ku70 甲基化和功能的调节
- 批准号:
10330477 - 财政年份:2021
- 资助金额:
$ 35.82万 - 项目类别:
Mechanisms of the BRCA-network in tumorigenesis and therapeutic response
BRCA 网络在肿瘤发生和治疗反应中的机制
- 批准号:
10599895 - 财政年份:2021
- 资助金额:
$ 35.82万 - 项目类别:
Project 4: The BRCA Network in Medulloblastoma Responses to Replication Stress
项目 4:髓母细胞瘤中 BRCA 网络对复制压力的反应
- 批准号:
10599907 - 财政年份:2021
- 资助金额:
$ 35.82万 - 项目类别:
Regulation of Ku70 methylation and functions by SETD4
SETD4 对 Ku70 甲基化和功能的调节
- 批准号:
10546482 - 财政年份:2021
- 资助金额:
$ 35.82万 - 项目类别:
Project 4: The BRCA Network in Medulloblastoma Responses to Replication Stress
项目 4:髓母细胞瘤中 BRCA 网络对复制压力的反应
- 批准号:
10396611 - 财政年份:2021
- 资助金额:
$ 35.82万 - 项目类别:
Mechanisms of the BRCA-network in tumorigenesis and therapeutic response
BRCA 网络在肿瘤发生和治疗反应中的机制
- 批准号:
10396606 - 财政年份:2021
- 资助金额:
$ 35.82万 - 项目类别:
Molecular modulators of radiation-induced chromosome instability and hematopoietic damage
辐射引起的染色体不稳定和造血损伤的分子调节剂
- 批准号:
10438851 - 财政年份:2015
- 资助金额:
$ 35.82万 - 项目类别:
Molecular modulators of radiation-induced chromosome instability and hematopoietic damage
辐射引起的染色体不稳定和造血损伤的分子调节剂
- 批准号:
10626749 - 财政年份:2015
- 资助金额:
$ 35.82万 - 项目类别: